The selection of cold-adapted mutants of selected human rotaviruses of major epidemiologic importance was reported previously. These mutants included strain D (VP4:1A; VP7:1), DS-1 (VP4:1B; VP7:2), Wa x DS-1 (VP4:1A; VP7:2), and Wa x P (VP4:1A; VP7:3). One of these (strain D) is currently under clinical evaluation as a potential vaccine candidate for use in humans. In an attempt to provide antigenic coverage for the fourth epidemiologically important VP7 serotype, VP7:4, we passaged a Wa x ST3 (VP4:1A; VP7:4) reassortant serially in primary African green monkey kidney cells at progressively lower suboptimal temperatures (30C, 28C, and 26C). Triple plaque purification of each mutant selected after tenth serial passages was performed at the temperature of that passage series. The ca and ts phenotypes of the plaque purified mutants were then examined. The 30C-derived mutant exhibited both the ca and ts phenotypes. The 28C-derived mutant exhibited an increase in temperature sensitivity, and the 26C-derived mutant exhibited a greater degree of temperature sensitivity in comparison to the corresponding ca 28 mutant.